In the interphase nucleus, DNA is necessarily organized around the histone core and, then, in higher order structures. At the electron microscope, dispersed euchromatin fibers and dark heterochromatin regions can be distinguished. The latter is divided in constitutive heterochromatin, reasonably localized at the periphery nearby the nuclear envelope and far from the perichromatin region where transcription occurs, and facultative heterochromatin, containing the genes undergoing silencing and activation (regulated genes) on the surface facing the perichromatin region. Chromatin structure affects numerous and fundamental genetic processes, such as gene expression, and therefore it is strictly regulated by different mechanisms. DNA methylation prevents the binding of transcriptional factors and recruits transcriptional repressor complexes, thus forming compact and inactive chromatin, although recent studies show 5-methylcytosine to reduce nucleosome stability. 5-methylcytosine was also found in the 3’ untranslated regions to probably influence mRNA stability. Histone post-transcriptional modifications can lead both to a more compact or relaxed chromatin structure by inducing nucleosome repositioning: for instance, H3K9me3 is known as a marker of constitutive heterochromatin while H3K27me3 or H4K20me3 label facultative heterochromatin. The ionic environment is also known to profoundly affect chromatin organization: an increase of calcium or magnesium results in different types of chromatin conformations, probably with different stability. Until now, DNA or RNA methylation, as well as histone modifications, was studied through biomolecular approaches. Here, we propose an alternative epigenetic analysis at ultrastructural level, using mouse hepatocytes to describe the distribution of 5-methylcytosine and histone modified residues on condensed chromatin regions and HeLa cells in order to detect epigenetic modifications on RNA fibrils. Treatments known to induce chromatin compaction (modification of cation concentration or heat shock) were also chosen to deeply investigate chromatin structure regulation. Therefore, cells and tissues were prepared principally for the cytochemical analysis, combining numerous immunocytochemical reactions with specific staining methods. This process is complex to analyze and more variables have to be considered. However, our results suggest that the main function of DNA methylation and histone modifications is the gene switching-on and -off, which occurs on chromatin surface, whereas they do not seem necessarily involved in the maintaining of chromatin condensation status, probably preserved by other mechanisms to be deeply investigated. As for RNA, our data indicate that cytosine methylation is a very precocious event, probably confirming its involvement in mRNA stability, and DNMT3A is unexpectedly involved in this modification. Other studies will be carried out to understand the processes regulating chromatin structure and the role of mRNA methylation. However, the influence of the ionic environment on both chromatin structure and the epigenetic modifications should be further studied.
|Titolo:||High-resolution epigenetic analysis of the cell nucleus|
|Data di pubblicazione:||18-gen-2018|
|Appare nelle tipologie:||8.01 Tesi di dottorato|